There is a critical need for better insulin replacement therapy to circumvent the complications of insulin-dependent diabetes mellitus (IDDM). Our goal is to develop techniques for transplantation of microencapsulated, xenogeneic islets to provide a durable, physiological source of insulin to diabetic patients. It has previously been shown that microcapsules are biocompatible and that xenogeneic islet grafts contained in microcapsules functioned indefinitely in the peritoneal cavity of mice with streptozotocin-induced (SZN) diabetes. Thus, microcapsules may be intact and stable in vivo and factors that may be required for long-term survival and function of the xenogeneic islets are accessible. The microcapsules serve as a mechanical barrier that prevents cell-to-cell contact between recipient lymphocytes and donor islets. The mechanical barrier primarily prevents host sensitization rather than protecting the graft from immune destruction, because encapsulated islets are very rapidly destroyed by recipients that are presensitized to the islet donor cell antigens. Similarly, encapsulated xenogeneic islets were rejected (in two weeks) by NOD mice, which is possibly due to presensitization of NODs to islet antigens. Xenografts undergoing rejection in NOD mice were surrounded by large numbers of activated macrophages and immunoglobulins, with IL-1α, TNFα, both documented by immunocytochemistry, and IL-4 messenger RNA detected by RT-PCR. We postulate that NOD rejection is initiated by donor antigens that are secreted by or shed from the encapsulated islets and which are processed via the MHC (major histocompatibility complex) class II pathway by host APC (antigen presenting cells). These APC activate NOD CD4+ T cells that develop into a Th2 response, with donor islet destruction occurring via cytokine-mediated events.
We have also been able to improve the microencapsulation process to permit long-term survival of concordant, rat islet xenografts, even in NOD mice. Furthermore, we have found that blockade of NOD co-stimulatory molecules with CTLA4Ig significantly prolongs survival of discordant, rabbit islet xenografts for up to 200 days. Thus, we have been able to overcome problems associated with transplanting encapsulated islet xenografts into autoimmune diabetic recipients.
Insulin-Dependent Diabetes Mellitus
The last several years have witnessed a remarkable increase in or knowledge of the effects of therapies for insulin-dependent diabetes mellitus (IDDM). The Diabetes Control and Complications Trial (DCCT) found that intensive insulin therapy delayed the onset and slowed progression of retinopathy, nephropathy, and neuropathy in patients with IDDM (1). Unfortunately, intensive insulin therapy is not appropriate for many IDDM patients; and even with careful monitoring, DCCT patients had increased episodes of severe hypoglycemia (1). Ironically, results of the DCCT support the rationale for pancreas and islet transplantation. Since the inception of islet transplant experiments, it has been the hope that such grafts might supply insulin more homeostatically than exogenous insulin can, and that ‘near-normal’ modulation of carbohydrate metabolism might prevent the secondary complications of IDDM (2). Clinical pancreas allografts have improved outcomes with the advent of combination immunosuppression; and near normal of glucose homeostasis follows most pancreatic allo- and auto-grafts (3). However, the first-year mortality of a human pancreatic allograft remains high (10%), immunosuppression is required, and only limited numbers of clinical whole-organ pancreatic transplants are being done worldwide (2, 4, 5).
The Rationale for Microencapsulated Islet Xenografts
Islet transplantation is an attractive therapy for patients with IDDM, since problems related to the exocrine pancreas may be avoided. However, allografts of donor human islets have not been successful long-term (3); and availability and yield of human islets are limited. Therapeutic islet transplants for large number of patients almost certainly will require donor islets harvested from animals (xenografts) (2, 4).
The optimal source of xenogeneic islets for clinical use remains controversial. Islets have been isolated from subhuman primates and xenografted into immunosuppressed, diabetic rodents, with short-term reversal of diabetes (6). However, there are significant ethical issues surrounding use of primates, Other promising sources are porcine, bovine, canine, and rabbit islets, which function remarkably well, (i.e., maintaining normoglycemia) in diabetic rodents until transplant rejection occurs (7–11). Long-term human, bovine and porcine islet xenograft survival has been documented in nude mice and rats, suggesting that sufficient islet-specific growth factors are present in xenogeneic recipients (2, 12–17). For sociologic/ethical reasons, canine islets are not clinically appropriate. Porcine islets are both difficult to isolate (intact) and to maintain in vitro; nevertheless, they are extremely promising for eventual clinical application (18–21). Isolation of bovine islets is technically easier (than porcine islets), and calf islets are glucose-responsive (22). Recently, large scale rabbit islets isolation has been developed (23) (see Preliminary Studies). Rabbit pancreas is an attractive source of islets. Rabbit, like porcine insulin, differs from human insulin at only one amino acid, and rabbit islets are glucose responsive (22, 24). In addition, most humans do not possess natural anti-rabbit antibodies, which might improve the possibility of preventing xenograft rejection (25). It is currently feasible to consider isolation of 1,000,000 donor islets/per human diabetic recipient from either calves, pigs or rabbits, utilizing multiple donors.
The most significant obstacle to islet xenotransplantation on human IDDM is the lack of an effective immunosuppressive regiment to prevent cross-species graft rejection (2, 26–28). Recently, it has been reported that human islets will survive long-term in SZN-diabetic mice treated either with anti-CD4 antibody (16) or CTLA4Ig (a high affinity fusion protein which blocks CD28-B7 interactions) (12), or by exposure of donor islets to purified high affinity anti-HLA (ab)2 (29). However, with the exception of these studies, indefinite survival of islet xenografts has rarely been achieved, except with the aid of porous, mechanical barriers. Both intra- and extra-vascular devices are under development. However, potential clinical complications, such as bleeding, coagulation, and bioincompatibility mitigate against their current use in diabetic patients (30, 31). For example, acrylic-copolymer hollow fibers placed subcutaneously maintained viability of human islet allografts for two weeks (50 islets per 1.5 cm fiber) (65,000 M.W. permeability) (32).
However, to implant 500,000 islet would require >150 meters of these hollow fibers, which is not clinically feasible.
One of the most promising islet envelopment methods is the polyamino acid-alginate microcapsule. A large number of recent studies have shown that intraperitoneal xenografts of encapsulated rat, dog, pig or human islets into streptozotocin-diabetic mice or rats promptly normalized blood glucose for 10–100+ days (7, 19, 33–39). Long-term normalization of hyperglycemia by microencapsulated canine islet allografts, porcine islet xenografts, and one human islet allograft has been reported (21, 40–42). The mechanisms by which microcapsules protect islet xenografts from host destruction are not fully understood. However, it has been suggested that prohibition of cell-cell contact with host immunocytes is important (30, 35). The marked prolongation of widely unrelated encapsulated islet xenografts in rodents with induced diabetes has prompted studies in animals with spontaneous diabetes.
The Spontaneously Diabetic NOD Mouse as a Model of Human IDDM
Nonobese diabetic (NOD) mice develop diabetes spontaneously, beginning at approximately twelve weeks of age. NOD mice are the most appropriate model for studying the feasibility of islet xenotransplants because their disease resembles human IDDM in several ways. Macrophage, dendritic cell and lymphocytic infiltration of islets can be detected as early as four weeks of age and precedes overt hyperglycemia (43–46). NOD diabetes is T lymphocyte-dependent (43–45); and it is associated with (MHC) Class II genes (47–50). Cytotoxic T cells and antibodies specific for beta cells or for insulin have been identified, characterized and cloned from NOD mice (44, 45, 51–55). Loss of tolerance to islet antigens in NODs correlates with appearance of Th1 immune responses to glutamic acid decarboxylase, a factor which has been reported to be a primary auto-antigen in human IDDM (5,657). The disease can be induced in non-diabetic, syngeneic mice by transfer of both CD8+ and CD4+ T cells or T-cell clones from diabetic NODs (44, 52, 55, 58); and inhibition of NOD macrophages or CD4+ T lymphocytes or treatment with anti-Class II monoclonal antibodies prevents or delays diabetes onset in NOD mice (59, 50). Defects in NOD macrophages, C5 complement and NK cell function have been reported (61). It has been suggested that helper T-cells function to activate CD8+ cells, which damage beta cells by direct cytotoxic attack. However, some recent studies have suggested that beta cell killing may be indirect, from a nonspecific inflammatory response which initially involves CD4+ cells, but also includes infiltrating macrophages, which release cytokines and oxygen free-radicals (particularly nitric oxide), known beta cell toxins (62–65). Because of similarities to IDDM, NOD mice are the best model in which to study islet xenografts.
Recently, the Scid mutation has been back-crossed onto the NOD background, resulting in immuno-deficient NOD-Scid mice (66–69). These mice homologous for the Scid mutation, which results in an inability to rearrange T-cell receptor and immunoglobulin genes (66, 67). The consequence is an absence of T and B-lymphocytes. These mice do not develop diabetes spontaneously; but they may be rendered diabetic with multiple low-dose streptozotocin (MLD-SZN) regimens, making them an optimal model for adoptive transfer experiments (67–69). NOD-Scids express NOD MHC genes and other genes that are relevant for development of the disease. They mount robust macrophage and limited NK-cell responses, but are functionally T- and B-lymphocyte deficient (69).
Islet Xenografts into Diabetic NOD Mice
Unlike mice with SZN-induced diabetes, diabetic NOD mice rapidly reject unencapsulated islet xenografts, allografts and isografts (7, 8, 10, 19, 33, 56, 70, 71). Conventional immunosuppressive regimens have little effect on this reaction (10, 71–73). Treatment of NOD recipients with monoclonal antibodies directed against CD4+ helper T lymphocytes or FK506 prolongs islet graft function (from 5 to 25 days) (7, 8, 10, 73); but long-term islet graft survival in NODs has not been reported.
Several laboratories have reported that intraperitoneal microencapsulated islets (allo- and xeno-geneic) function significantly longer than non-encapsulated controls, but eventually are destroyed also by recipients with spontaneous (autoimmune) diabetes (NOD mice or BB rats) (7, 9, 19, 33, 35, 70, 74–78). Rejection is accompanied by an intense cellular reaction, composed primarily of macrophages and lymphocytes, which entraps islet-containing microcapsules and recurrence of hyperglycemia within 21 days, in both NOD and BB recipients (7, 19, 74, 76, 77). The mechanism of encapsulated islet rejection by animals with spontaneous diabetes remains incompletely understood, but the fact that it rarely occurs in mice with induced (SZN) diabetes suggests that anti-islet autoimmunity may be involved in islet graft destruction.
Mechanisms of NOD Destruction of Encapsulated Islet Xenografts: Macrophages, T-Cells, and Cytokines
It has been suggested by several investigators that microcapsules, like other bioartificial membrane devices promote survival of xenogeneic and allogeneic islets by: (A) preventing or minimizing release of donor antigen(s), thereby reducing host sensitization, and/or (B) preventing or reducing host effector mechanisms (i.e. T-cell contact, anti-graft antibody binding, cytokine release).
Most studies of rejection of islets in microcapsules and other membrane devices have focused on effector mechanisms. For example, Halle (35) and Darquy and Reach (79) reported that microcapsules protected donor islets from host immunoglobulins, specifically human anti-islet antibodies and complement effects, in vitro. Although complement components, are too large (>>150,000 Kd) to enter conventional poly-l-lysine microcapsules, it is possible that antibodies combine with shed donor antigens forming complexes which bind to FcR of macrophages in vivo (in the peritoneal cavity) which could initiate cytokine release causing encapsulated islet destruction (80). Complement could facilitate binding of complexes to macrophages via the C3b receptor or by the release of chemotactic peptides that could increase the number of macrophages.
Involvement of NOD T-lymphocytes in rejection of encapsulated islets has been proposed by Iwata, et al. (81), who found significant prolongation of encapsulated hamster-to-NOD mouse encapsulated islet xenografts when NOD recipients were treated with deoxyspergualin (DSG), a T-cell inhibitory immunosuppressant (81). This data is consistent with prior finding of several laboratories, that treatment of NODs with monoclonal antibodies directed against CD4+ helper T cells or FK-506 prolonged function of both encapsulated and nonencapsulated rat-to-NOD islet xenograft (7, 8, 10, 73) and these finding are similar to observations of Auchincloss (27), Pierson (82) and Gill (83), that CD4+ T cells play a dominate role in xenoreactivity.
A prominence of macrophages/monocytes in peri-microcapsular infiltrates of encapsulated islet allografts and xenografts in NOD mice and BB rats has been reported (7, 33, 36, 74, 76–78, 84). Cytokines known to be products of macrophages, including IL-1 and TNF (62, 77, 85, 86), may be involved destruction of encapsulated islets. Both IL-1 and TNF have been reported to reduce insulin secretion and cause progressive damage of islet cells in vitro (58, 62–64, 85–87). Cytokine-mediated injury might occur directly or indirectly, by activation of an intraperitoneal inflammatory response (30, 77). Recently, it has been reported by Dr. J. Corbett (IPITA conf. June 1995), that there are as many as ten macrophages within each islet. IL-1 induces nitric oxide synthase (NOS) (63–65), with resultant generation of nitric oxide (NO), which causes injury to mitochondria and to DNA in beta cells (63–65). Furthermore, this pathway of islet damage is worsened by TNF (88, 89). Theoretically, macrophages from within donor islets and host peritoneal cavity or within the down islets could be involved in cytokine-mediated damage to encapsulated islets.
Studies of cytokine messenger RNA profiles in hamster-to-rat liver and pig-to-mouse islet xenografts have found selective increases in Th2 cytokines (IL-4, IL-5, IL-10) and no change from normal in IL-2 (11, 90). These are distinctly different from those of O'Connell, et al. (91, 92), who reported IL-2 messenger RNA in biopsies of allograft rejections of nonencapsulated islets. Increased Th2 activity relative to Th1 (93–95) activity is distinct from the known NOD ‘Th1’ anti-islet immune response (56, 57, 96). The Th2 response is characteristic of evoked antibody responses to foreign antigens and suggests that humoral reactions to encapsulated xenografts may be of critical importance. Furthermore, strategies designed to abrogate ‘Th2’ responses may significantly prolong encapsulated islet xenograft survival. The ‘Th2’ helper T-cell cytokine mRNA profile is characteristic of antibody responses to foreign antigens.
Costimulatory Molecules, APC's and Islet Xenograft Destruction by NOD Mice
Involvement of APCs in immune responses to islet xenografts is suggested by recent studies of Lenschow, et al. (12), who found that blockade of the co-stimulatory molecule, B7 with the soluble fusion protein, CTLA4Ig, prolonged human-to-mouse islet xenografts in SZN-diabetic mice. Several studies, in vitro and in vivo, have shown that foreign molecules which interact with the T cell receptor (peptides, specific antibodies, mitogens) fail on their own to stimulate naive T cells to proliferate (95, 97), and may induce antigen-specific anergy. At least one additional (costimulatory) signal is required, and it is delivered by APCs. In mice, one such costimulatory pathway involves the interaction of the T-cell surface antigen, CD28 with either one of two ligand, B7-1 and B7-2, on the APCs (95, 97–102). Once this full interaction of T-cells and APCs occurs, however, subsequent re-exposure of T-cells to peptide, mitogen, etc. will result in proliferation in the absence of costimulation. (95).
CTLA4 is a cell surface protein that is closely related to CD28; however, unlike CD28, CTLA4 is expressed only on activated T-cells. B7-1 has a high affinity for CLTA4 than CD28; and it has been suggested that CTLA4 may modulate functions of CD28 (97, 103, 104). CTLA4Ig is a recombinant soluble fusion protein, combining the extracellular binding domain of the CTLA4 molecule with constant region of the IgG1 gene. Both human and murine CTLA4Ig have been shown to inhibit T-lymphocyte responses in mice (141, 142). Administration of CTLA4Ig to mice has been shown to induce antigen-specific unresponsiveness (in a murine lupus model) (97, 99, 105) and long-term acceptance of murine cardiac allografts (106, 107). In addition, Lenschow, et al., found that it induced tolerance to human islets in SZN-diabetic mice (12). CTLA4Ig has also been reported to reduce the incidence of diabetes in NODs (108). There are no reports of effects of CTLA4Ig on islet graft survival in spontaneously-diabetic recipients, such as NOD mice. However, our studies show that CTLA4Ig significantly prolongs survival of encapsulated rabbit islets in NOD recipients.
Recent studies have further illuminated helper T-cell-APC interactions, with recognition of the importance of binding of the APC-CD40 antigen to its ligand, GP39, on helper T-cells (109, 110). A monoclonal hamster anti-murine GP39 antibody (MR1) blocks helper T-cell interactions with APCs, macrophages, effector T-cells and B-lymphocytes (109, 110). Dr. A. Rossini has reported recently (IPITA conf. June 1995) that MR1 plus B7 negative donor spleen cells day 7 allows long-term survival of both allo- and xeno-geneic islets in SZN-diabetic mice.
The Immunogenicity of Encapsulated Islets and Mechanisms of Graft of Destruction
Empty microcapsules have been reported to elicit no cellular responses (33, 35, 36). On the other hand, others have found reactions to empty capsules, (30, 76, 77, 111, 112). Impurities in reagents such as contamination with endotoxin or high concentrations of mannuronate most likely contribute to bioincompatibility (113). It is apparent that some formulations of poly-l-lysine microcapsules are biocompatible and some are not. Until standardized reagents are available, immunologic studies are microencapsulated islets can only be interpreted when investigators include empty microcapsule controls which document their biocompatibility.
Recently, de Vos, et al. (114) reported incomplete encapsulation or actual protrusion of islets through microcapsule membranes in some microcapsules, and suggested this biomechanical imperfection is one factor in microcapsule destruction. Similar observations have been made by Chang (115), who found incorporation of islets and hepatocytes within the walls of poly-l-lysine alginate microcapsules. Several other investigators have published photomicrographs of encapsulated islets showing obvious entrapment of islets in capsules, walls, but did not comment on this problem (35, 116, 117). Incomplete encapsulation would be anticipated to result in premature capsule fracture and exposure of donor islets to host cells; but there are no reports analyzing this as a source of donor antigen exposure, sensitization and host.
Relatively few studies have focused on the role of donor islet antigen(s) released from microcapsules in initiating host immune responses. Ricker, et al. (33) reported similar, intense cellular reactions by NOD mice to rat insulinoma, hepatoma and pheochromocytoma cell lines in microcapsules and concluded that the NOD immune reaction was not islet-specific. Horcher, et al. (36) reported 15-week survival of 6/7 encapsulated Lewis rat islet isografts, compared to failure of 8/10 encapsulated Wistar-to-Lewis islet allografts within 56 days. Isograft biopsies showed viable islets, intact capsules and no pericapsular immune reaction (36), while biopsies of failed allografts revealed pericapsular cellular responses and nonviable islets. This is the only report in the literature with encapsulated islet isograft controls. Although the Lewis rat model is not one with autoimmune diabetes, the results are significant, and suggest that donor antigen(s) are the stimulus for subsequent host responses.